1. Field of the Invention
The present invention relates to a novel phosphorescent compound and to a method of use of such phosphorescent compound as a label or marker for binding with immunoglobulins or chemical analytes. It specifically relates to palladium (II) octaethylporphine alpha-isothiocyanate as a novel phosphorescent compound.
2. Description of the related art
Generally, the assays of the art have included radioimmunoassays for insulin and other hormones. The radioisotopes attach to hormones which have been chemically modified and then mixed with non-radioisotope labeled hormone. This admixed solution is contacted with a limited amount of antibody which is specific for the hormone. After separating the bound antibody from the unbound a determination can be made as to the concentration of the hormone in the patient's blood by comparing the radioactivity level of the patient's hormone to the radioactivities of known concentrations of hormones.
In other areas of the art, biological assays have provided a valuable tool in the evaluation of the effect of pharmacologically active compounds including biologically active proteins. Procedures developed for the bioassay of these types are usually biphasic. One procedure incubates the substrate cell population with the substance to be tested. The art has developed widely divergent procedures to carry out the quantitative analysis of the cellular response to the sample compound.
To obtain the quantitative analysis the art includes immunomediated assays which are quantitative only for the binding of the molecule to the cell. Another type of assay requires cell staining and visual or microscopic observation of a change in cellular morphology. Such assays are dependent on a subjective evaluation of the results.
The use of radioactive metabolites to determine viability of an antibody is less subjective than the colorimetric systems but the processing of numerous samples is time consuming, labor intensive and expensive. Further, counting of assay cell with the aid of an electronic particle counter to determine cell growth requires large multiples of each sample for accurate results and does not truly reflect the viability of the assay cells.
Even more recently, the art has developed colorimetric analysis of cell growth either by direct staining of the cell monolayer or by cellular dye uptake. For example, dye molecules have been used as labels for proteins (e.g. antibodies) to make reagents which have been associated with fluorescence readings. However, it is known in the art that labeling an antibody with a dye molecule and measuring the absorbance value does not provide a level of sensitivity sufficient to enable an accurate quantitative measurement of ligands in immunoassay systems.
Thus, the present state of the art indicates that although radioimmunoassays have been the cornerstone of modern immunoassay procedures, non-radioisotopic methods such as enzyme-linked immunosorbent assays and fluorescent immunoassays have become predominant in clinical laboratories due to aforementioned shortcomings of the radioimmunoassays and due to the licensing, special skills, expensive equipment, shipping and disposal problems associated with radioisotopes.
However, the present state of the art still has objections and has lack of sufficient sensitivity for immunoassay procedures to achieve full acceptability in the art. The non-radioisotope immunoassays generally lack the ability to detect molecular concentrations to the extent that radioimmunoassays can measure in large part due to the light scatter and other types of interference inherent in the testing procedure. Enzyme-linked immunosorbent assays require bulky, labile enzymes, are susceptible to inhibition and denaturation, and an additional incubation step is needed with a substrate to monitor the enzyme's activity.
It is recognized that fluorescence immunoassays achieve greater levels of sensitivity than enzyme-linked immunosorbent assays due to fluorescence molecules, i.e., the fluorophore labels, absorbing energy at one wavelength, i.e., excitation, and radiating energy at another wavelength, i.e., emission. This difference in wavelengths, known as the Stokes shift, can be used advantageously to gain sensitivity by having the excitation light project at 90.degree. to the emission light being detected by a photomultiplier tube or other known detection devices. It is also recognized that each of the materials used as a fluorescent label has its own characteristics of required wavelength of excitation and resulting wavelength of fluorescent emission, i.e., has its own Stokes shift. If the label has a low Stokes shift, it is difficult and expensive to design light sensors which will respond to the wavelength of fluorescence and be relatively insensitive to the wavelength of the excitation light. Such sensors, as mentioned, use expensive defraction gratings inserted between the sample containing the fluorescent labels and the optical sensor. These are placed so that they are orthogonal to the direction of a beam of light at the excitation wavelength. There is still some background scatter and noise to contend with due to soluble molecules, small colloidal particles, or the presence of solid-phase material.
Thus, there is a need for a better luminescent compound for use in immunoassays, but the prior art considered as a whole, neither teaches nor suggests how the prior art compounds, if any, might be improved.